Non-invasive molecular imaging is a powerful technique aimed at tracking cellular and molecular events in their native environment in the intact living subject. In its broadest sense, molecular imaging entails the administration of a tracer molecule labeled with a contrast reagent for visualization. Primarily, radioactively labeled tracers are used in combination with positron-emission tomography (PET) or single photon emission-computed tomography (SPECT)-based imaging techniques (Pysz et al. 2010, Clin. Radiol. 65:500-16). In the clinic, the majority of cancer imaging is currently still performed based on detection of enhanced metabolism in cancer cells using 18F radiolabeled deoxyglucose (Coenen et al. 2010, Nucl. Med. Biol. 37:727-40), while 99mTc-labeled human serum albumin is used for lymphoscintigraphic mapping of the draining lymph nodes in cancer (Kim et al. 2001, Int. J. Oncol. 19:991-6). Although useful, these tracers do not target a specific molecule or receptor on the surface of the cells involved in the disease process. Therefore, there is a need for probes that allow a more specific molecular characterization of inflamed or diseased tissue using disease related membrane antigens. These specific markers can help to define the phenotype of a disease and can be targeted by specific agents like monoclonal antibodies (MAbs). In this context, the choice of the targeted molecular markers will be a critical factor in determining whether it is possible to acquire in-depth molecular information on the underlying disease process.
Several FDA approved MAbs directed against tumor-associated antigens (TAAs) on malignant cells are being applied for diagnosis and treatment of cancer, with a few of the most commonly used MAbs being human epidermal growth factor receptor 2 (HER2)-specific Trastuzumab (Dijkers et al. 2010, Clin. Pharmacol. Ther. 87:586-92), carcinoembryonic antigen (CEA)-specific Arcitumomab (Hong et al. 2008, Biomark Insights 3:435-451) and prostate-specific membrane antigen (PSMA)-specific Capromab (Aparici et al. 2012, Am. Nucl. Med. Mol. Imaging 2:48-54). Yet, although the direct targeting of antibody moieties to TAAs on malignant cells is a potent tool that has reached clinical maturity, the non-transformed cells present within the tumor microenvironment can also provide useful biomarkers for molecular imaging, as an alternative or complement to markers on the inherently genetically instable transformed cells. Indeed, tumors should be considered as organ-like structures featuring a complex bidirectional interplay between transformed (cancer) and non-transformed (stromal) cells, whereby stromal cells can critically contribute to tumor initiation, growth and metastasis. Hence, targeting these tumor-associated stroma cells for imaging could provide additional information on the state of the tumor or response to therapy.
In particular, tumor-associated macrophages (TAMs) are an important component of the tumor stroma, both in murine models and human patients (Pollard 2004, Nat. Rev. Cancer 4:71-8). TAMs can promote tumor-growth by affecting angiogenesis, immune suppression and invasion and metastasis (Lin et al. 2006, Cancer Res. 66:11238-46). The plasticity of macrophages offers perspectives for using them as in vivo sensors for the tumor microenvironment they are exposed to. As a matter of fact, at the tumor site, these cells are confronted with different tumor microenvironments, leading to different TAM subsets with specialized functions and distinct molecular profiles (Laoui et al. 2011, Int. J. Dev. Biol. 55:861-867). For example, in mammary tumors, at least two distinct TAM subpopulations have been described, based on a differential expression of markers such as the macrophage mannose receptor (MMR or MHC II), differences in pro-angiogenic or immunosuppressive properties and intratumoral localization (normoxic/perivascular tumor areas versus hypoxic regions). In particular, the association of MMR-high TAMs with hypoxic regions in the tumor (Movahedi et al. 2010, Cancer Res. 70:5728-5739) offers perspectives for image-guided radiotherapy.
Full-sized MAbs have a number of disadvantages that have so far limited their effective use in the clinic. MAbs are macromolecules with a relatively poor penetration into solid and isolated tissues such as tumors (Hughes et al. 2000, J. Clin. Oncol. 18:363-370). In addition, complete MAbs feature a long residence time in the body and a potential increase in background signals because of binding to Fc receptors on non-target cells, making them less suitable for molecular imaging applications. Indeed, for imaging the most important properties of a tracer are: rapid interaction with the target, fast clearing of unbound molecules from the body and low non-specific accumulation, especially around the area of interest. These requirements have led to the development of a myriad of antibody derived probe formats, like Fabs and scFvs, trying to combine specificity with a small size for favorable pharmacokinetics (Kaur et al. 2012, Cancer Lett. 315:97-111).
A novel approach for generating small and high-affinity antigen-binding moieties focuses on the use of single-domain VHH antibody fragments, named NANOBODIES®, derived from the heavy-chain only antibodies found in camelid species (Hamers-Casterman et al. 2003, Nature 363:446-448). sdAbs, conveniently labeled with 99mTc at their carboxy-terminal hexahistidine-tail, have by now a solid track record for SPECT-based molecular imaging in preclinical animal models (reviewed in: Vaneycken et al. 2011, Curr. Opin. Biotechnol. 22:877-881), with rapid blood clearance of unbound probes and high signal-to-noise ratios as early as a few hours after inoculation. In particular, US2011/0262348 demonstrates the usefulness of 99mTc-labeled mouse-specific anti-MMR sdAbs for targeting MMR-positive TAMs in mice models that spontaneously develop carcinomas. These results offer perspectives for applications of anti-MMR sdAbs in image-guided radiotherapy, whereby the distribution of radiation is adapted in function of localized risk factors such as hypoxia (Bentzen 2005, Lancet Oncol. 6:112-117). Moreover, as has been documented for sdAbs targeting the HER2 tumor antigen, sdAbs exhibiting effective tumor targeting can be converted from an imaging probe in a radioimmunotherapeutic compound by coupling it to a therapeutic radionuclide (D'Huyvetter et al. 2012, Contrast Media Mol. Imaging 7:254-264).
However, there is still a need for specific probes that can be used both in the clinic and in preclinical animal models, with applications including improved diagnosis, prognosis, treatment and therapy monitoring.